Proteomics Analysis Of Serous Ovarian Carcinoma

Serous ovarian carcinoma is the most common subtype of epithelial ovarian cancer which remains the leading cause of death from gynecologic malignancy. The pathogenesis of serous ovarian carcinoma remains largely unknown. Recently, the viewpoint that malignancy is a kind of molecular disease is generally accepted. Authors believe that altered expression of certain clusters of genes/proteins probably contribute to the gain and maintain of specific features of malignant tumors. Quantitative changes in protein expression but not gene expression can eventually reflect the phenotypic biologic properties of ovarian cancer. Although a large number of studies have demonstrated various molecules including genes and proteins associated with the development or progress of cancers, few of them are recognized to be specific for epithelial ovarian cancer. In this study, we applied proteomic techniques to analyze the protein expression profiles of serous ovarian carcinoma and normal ovarian epithelium tissue aiming to characterize tumor-specific changes in the proteome of serous ovarian cancer, which may bring out new valuable diagnostic biomarkers and/or promising therapeutic targets for serous ovarian cancer, and further more, may provide new insight into carcinogenesis of serous ovarian caner. Using two-dimensional electrophoresis and silver staining, combined with PDQUEST analysis, approximately 1800 proteins spots were detected in normal ovarian epithelium and serous ovarian carcinoma tissue groups. Compared with the normal control group,63 protein spots exhibited significantly differential expression, including 38 up-regulated spots and 25 down-regulated spots.60 of them were successfully identified by MALDI-TOF/TOF MS, representing 54 unambiguous and unique proteins. To further confirm the protein alterations in SOC revealed by proteomic analysis, BANF1, galectin-1, GMFB, GGCT, HDGF, and RhoGDI 2 were selected for validation. The expression changes of these selected proteins were consistent with the 2-DE and silver-staining results, which validated that the differential expressions of proteins obtained from proteomic analysis were convincing. Corresponding gene expression analysis of these proteins was also performed using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The levels of ARHGDIB and HDGF mRNA expression were upregulated and those of BANF1 and LAGLS1 mRNA were downregulated in serous ovarian carcinoma compared with normal epithelium. But no changes of GMFB and GGCT mRNA expression were found in serous ovarian carcinoma, compared with its normal counterpart, which was not consistent with the alterations of protein expression.Additionally, we analyzed glia maturation factor beta (GMFB) protein expression by immunohistochemistry in 246 patients with various degrees of ovarian epithelial lesions including 45 normal ovarian epithelia,51 benign ovarian serous adenomas,40 borderline ovarian serous adenomas, and 110 serous ovarian carcinomas. GMFB expression was found to be gradually elevated from normal epithelium, to benign serous adenoma, to borderline serous adenoma, to serous ovarian carcinoma tissues, and was positively correlated with FIGO stage (P=0.012). High GMFB expression was associated with poor disease-free survival (P=0.010) and overall survival (P=0.003), while multivariate analysis revealed GMFB to be an independent prognostic factor for disease-free survival (P=0.026) and overall survival (P=0.006) in patients with SOC. Using RNAi technique, GMFB expression in SKOV3 cell line was silenced and the cells consequently exhibited significantly decreased proliferation (P＜0.05).We therefore propose that proteins identified here may be involved in the development or progression of serous ovarian carcinoma, and GMFB can be considered as a prognostic predictor for SOC patients, as well as a potential promising therapy target. PART I Identification of differentially expressed proteins between normal ovarian epithelium and serous ovarian carcinoma tissuesObjective:To identify the differentially expressed proteins between normal ovarian epithelium and serous ovarian carcinoma tissues.Methods:Total proteins were extracted from 10 cases of serous ovarian carcinoma and 10 cases of normal ovarian epithelia. The proteins were separated using Two-dimensional electrophoresis and the gels were subjected to modified silver staining method compatible with MS. The stained gels were scanned using the high-resolution scanner GS-800 calibrated densitometer followed by analysis with PDQUEST analysis. Student's t-test statistical analysis with 99% significance level has been applied to the replicate groups. Significantly differentially expressed protein spots match the threshold (>2 fold) and statistical analysis standard were selected.Results:Approximately 1800 proteins spots were detected in both groups (Figure 1 A and 1B). Compared with the normal control group,63 protein spots exhibited significantly differential expression, including 38 up-regulated spots and 25 down-regulated spots.60 spots were successfully identified by MALDI-TOF/TOFMS, representing 54 unambiguous and unique proteins.Conclusions:1,The differentially expressed proteins identified here may involve in the carcinogenesis of serous ovarian carcinoma.2,Proteome technique is fairly powerful for the detection of tumor-specific proteins.PART II Validation of differentially expressed proteins and quantization of corresponding gene expression level of selected proteins.Objective:To further confirm the protein alterations in serous ovarian carcinoma revealed by proteomic analysis and to compare protein expression with their corresponding mRNA expression.Methods:Six proteins of interest, BANF1, galectin-1, GMFB, GGCT, HDGF, and RhoGDI 2 were selected. Total proteins and RNA were extracted from 20 cases of serous ovarian carcinoma and 20 cases of normal ovarian epithelia. Protein expressions were examined using western blot and gene expression levels were quantified employing real time RT-PCR.Results:GMFB, GGCT, HDGF, and RhoGDI 2 protein expression were upregulated in serous ovarian carcinoma; BANF1, galectin-1 protein expression were decreased in serous ovarian carcinoma; HDGF, and ARHGDIB mRNA expression were elevated in serous ovarian carcinoma; BANF1, galectin-1 mRNA expression were downregualted in serous ovarian carcinoma; no changes of GMFB and GGCT mRNA expression were found in serous ovarian carcinoma. Conclusions:1,The expression changes of these selected proteins were consistent with the 2-DE and silver-staining results, which validated that the differential expressions of proteins obtained from proteomic analysis were convincing.2,GMFB and GGCT mRNA expression alterations were not consistant with that of proteins, suggesting that the expressions of the two proteins are likely to be influenced by translation and post-translational mechanisms in these tissues.PARTⅢGMFB expression in various serous ovarian epithelial lesions and survival analysisObjective:To analyze GMFB protein expression in various degrees of ovarian epithelial lesions and to determine the correlation between GMFB protein expression with clinicopathologic features and survival of patients with serous ovarian carcinoma.Methods:Employing immunohistochemistry to detect GMFB expression in 45 normal ovarian epithelia,51 benign ovarian serous adenomas,40 borderline ovarian serous adenomas, and 110 serous ovarian carcinomas; Collection the clinicopathologic features and survival data of these patients with serous ovarian carcinoma; using diverse statistical processes to analyze the results. Results:GMFB expression was found to be gradually elevated from normal epithelium, to benign serous adenoma, to borderline serous adenoma, to serous ovarian carcinoma tissues, and was positively correlated with FIGO stage. High GMFB expression was associated with poor disease-free survival and overall survival, while multivariate analysis revealed GMFB to be an independent prognostic factor for disease-free survival and overall survival in patients with SOC.Conculsions:1,GMFB may contribute to the initiation and development of serous ovarian carcinoma.2,GMFB can be considered as a prognostic predictor for SOC patients, as well as a potential promising therapy target.PART IV The effect of GMFB expression upon proliferation of SKOV3 cell lineObjective:To investigate the effect of GMFB expression upon proliferation of SKOV3 cell line.Methods:Using sequencing-targeting siRNA to silence GMFB expression in SKOV3 cell line and cellular proliferation was detected. Results:GMFB expression silenced cells showed decreased celluar proliferation.Conclusions:GMFB may contribute to the progression of serous ovarian carcinoma due to promotion of cellular proliferation.